Figures Legend
Fig 1. Screening of base editing in cells. (A) Architecture of cytosine base editor 4 (BE4). (B) Replacement of Cas9 in pX330 with EGFP. (C) Plasmid source of EGFP. (D) Fluorescent imaging of transfected HEK293 cell stably expressing EGFP. Scale bar, 100 μm. (E) Relative fluorescence intensity of D. (F) Base change in EGFP by sequencing. (G) Premature stop codon targeting of Dip2a and Dip2c genes in B16 cells. PAM sequences labeled in green, wild type base in blue, mutated base in red and stop codon underlined. (H) Western blot analysis. β-actin served as a loading control. (I) Efficiency of C>T base editing.P- value was determined by t -test. *P<0.05, ***P<0.001.
Fig 2. BE4 mediated C>T base editing in mice. (A) Schematic of sgRNA design at Tyr locus. (B) Working model of base editing in mice. (C) Schematic depiction of BE4 base editing. (D) Coat color of 8 day old Tyr mutant founders with mosaic pigmentation. (E) Chromatograms of WT and mutant sequences showing C>T substitution.
Fig 3. Screening of mutations in mice by genomic PCR and sequencing. (A) Sequences of Tyr gene target region in exon 1. SgRNA target sequence in blue and PCR primer sequences in green and orange. (B) Genomic PCR of target regions of founders 1-16 (F0). (C) Alignments of major genomic sequence signals from all founders. C-to-T base substitution is shown in red and green. Wild type in blue. (D) Frequencies of C>T base editing. (E) Chromatograms showing sequencing signals of PCR amplified region. Red arrow shows the base change. P- value was determined based on t -test. ****P<0.0001.
Fig 4. Chromatogram sequencing analysis of potential off-target sites (POTs) for sgRNAs predicted according to the online platform (https://benchling.com/)